The nutritional requirements for acetoin production by Bacillus subtilis CICC 10025 were optimized statistically in shake flask experiments using indigenous agroindustrial by-products. The medium components considered for initial screening in a Plackett-Burman design comprised a-molasses (molasses submitted to acidification pretreatment), soybean meal hydrolysate (SMH), KH2PO4 center dot 3H(2)O, sodium acetate, MgSO4 center dot 7H(2)O, FeCl2, and MnCl2, in which the first two were identified as significantly (at the 99% significant level) influencing acetoin production. Response surface methodology was applied to determine the mutual interactions between these two components and optimal levels for acetoin production. In flask fermentations, 37.9 g l(-1) acetoin was repeatedly achieved using the optimized concentrations of a-molasses and SMH [22.0% (v/v) and 27.8% (v/v), respectively]. a-Molasses and SMH were demonstrated to be more productive than pure sucrose and yeast extract plus peptone, respectively, in acetoin fermentation. In a 5-l fermenter, 35.4 g l(-1) of acetoin could be obtained after 56.4 h of cultivation. To our knowledge, these results, i.e., acetoin yields in flask or fermenter fermentations, were new records on acetoin fermentation by B. subtilis.